EP3105338B1 - Procédé de production d'un acide l-aminé au moyen d'une bactérie de la famille des enterobacteriaceae dans laquelle le gène yajl est surexprimé - Google Patents

Procédé de production d'un acide l-aminé au moyen d'une bactérie de la famille des enterobacteriaceae dans laquelle le gène yajl est surexprimé Download PDF

Info

Publication number
EP3105338B1
EP3105338B1 EP15708898.0A EP15708898A EP3105338B1 EP 3105338 B1 EP3105338 B1 EP 3105338B1 EP 15708898 A EP15708898 A EP 15708898A EP 3105338 B1 EP3105338 B1 EP 3105338B1
Authority
EP
European Patent Office
Prior art keywords
gene
coli
yajl
amino acid
strain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP15708898.0A
Other languages
German (de)
English (en)
Other versions
EP3105338A1 (fr
Inventor
Valery Vasilievich SAMSONOV
Natalia Sergeevna Eremina
Natalia Viktorovna Stoynova
Vera Georgievna Doroshenko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ajinomoto Co Inc
Original Assignee
Ajinomoto Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ajinomoto Co Inc filed Critical Ajinomoto Co Inc
Publication of EP3105338A1 publication Critical patent/EP3105338A1/fr
Application granted granted Critical
Publication of EP3105338B1 publication Critical patent/EP3105338B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/20—Aspartic acid; Asparagine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)

Definitions

  • the present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids by fermentation of a bacterium of the family Enterobacteriaceae which has been modified to overexpress the yajL gene.
  • L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
  • Another method for enhancing L-amino acid production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, genes which divert the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of the carbon, nitrogen, and phosphate fluxes, and genes encoding toxins.
  • the yajL gene encodes the YajL protein which belongs to the PfpI/Hsp31/DJ-1 superfamily that includes chaperones, peptidases, and the protein DJ-1 ( Messaoudi N. et al., Global stress response in a prokaryotic model of DJ-1-associated Parkinsonism, J. Bacteriol., 2013, 195(6):1167-1178 ).
  • the YajL is the closest prokaryotic homolog of DJ-1.
  • the Escherichia coli E.
  • YajL protein has 40% sequence identity and a similar three-dimensional structure with human DJ-1, an oncogene and neuroprotective protein whose loss-of-function mutants are associated with certain types of familial, autosomal recessive Parkinsonism ( Wilson M.A. et al., The atomic resolution crystal structure of the YajL (ThiJ) protein from Escherichia coli: a close prokaryotic homologue of the Parkinsonism-associated protein DJ-1, J. Mol. Biol., 2005, 353(3):678-691 ). The high homology and similarity of crystal structures of YajL and DJ-1 suggest that the proteins have similar function.
  • YajL protects bacteria against oxidative stress and oxidative-stress-induced protein aggregation possibly through its chaperone function and control of gene expression ( Kthiri F. et al., Protein aggregation in a mutant deficient in YajL, the bacterial homolog of the Parkinsonism-associated protein DJ-1, J. Biol. Chem., 2010, 285:10328-10336 ).
  • YajL functions as a covalent chaperone that, upon oxidative stress, forms mixed disulfides with chaperones, proteases, ribosomal proteins, catalases, peroxidases, and FeS proteins ( Messaoudi N. et al., J. Bacterial., 2013, 195(6):1167-1178 ).
  • An aspect of the present invention is to provide a method for producing an L-amino acid such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine using a bacterium of the family Enterobacteriaceae as described hereinafter.
  • L-amino acid such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine,
  • L-amino acids such as, in particular, but not limited to, L-amino acids of the pyruvate family such as L-valine, and aromatic L-amino acids such as L-phenylalanine.
  • An aspect of the present invention is to provide a method for producing an L-amino acid comprising:
  • L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, L-tyrosine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine.
  • L-amino acid is selected from the group consisting of L-alanine, L-isoleucine, L-leucine, L-valine and L-phenylalanine.
  • an L-amino acid-producing bacterium can mean a bacterium of the family Enterobacteriaceae which has an ability to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium or the bacterial cells when the bacterium is cultured in the medium.
  • an L-amino acid-producing bacterium can also mean a bacterium which is able to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain, such as E. coli K-12, and can mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.5 g/L or not less than 1.0 g/L of the target L-amino acid.
  • L-amino acid-producing ability can mean the ability of the bacterium to produce, excrete or secrete, and/or cause accumulation of the L-amino acid in a medium or the bacterial cells to such a level that the L-amino acid can be collected from the medium or the bacterial cells, when the bacterium is cultured in the medium.
  • L-amino acid can mean L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
  • aromatic L-amino acid includes, for example, L-phenylalanine, L-tryptophan, and L-tyrosine.
  • L-histidine has an aromatic moiety such as an imidazole ring
  • aromatic L-amino acid can also include, besides the aforementioned aromatic L-amino acids, L-histidine.
  • non-aromatic L-amino acid includes, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine.
  • non-aromatic L-amino acid can also include, besides the aforementioned non-aromatic L-amino acids, L-histidine.
  • L-amino acid can belong to one or more L-amino acid families.
  • the L-amino acid can belong to the glutamate family including L-arginine, L-glutamic acid, L-glutamine, and L-proline; the serine family including L-cysteine, glycine, and L-serine; the aspartate family including L-asparagine, L-aspartic acid, L-isoleucine, L-lysine, L-methionine, and L-threonine; the pyruvate family including L-alanine, L-isoleucine, L-valine, and L-leucine; and the aromatic family including L-phenylalanine, L-tryptophan, and L-tyrosine.
  • L-amino acids can be the intermediate amino acids in a biosynthetic pathway of a particular L-amino acid
  • the aforementioned families of amino acids may also include other L-amino acids, for example, non-proteinogenic L-amino acids.
  • L-citrulline and L-ornithine are amino acids from the arginine biosynthetic pathway. Therefore, the glutamate family may include L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline
  • L-Arginine, L-cysteine, L-glutamic acid, L-histidine, L-isoleucine, L-lysine, L-ornithine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, and L-valine are particular examples.
  • the pyruvate family amino acids such as L-alanine, L-isoleucine, L-valine, and L-leucine are preferable examples.
  • the aromatic amino acids such as L-phenylalanine, L-tryptophan, and L-tyrosine are yet preferable examples.
  • L-valine and L-phenylalanine are more preferable examples.
  • L-amino acid includes not only an L-amino acid in a free form, but may also include a salt or a hydrate of the L-amino acid, or an adduct formed by the L-amino acid and another organic or inorganic compound.
  • E. coli which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, those described in the work of Neidhardt et al. can be used ( Bachmann, B.J., Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In F.C. Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C., 1996 ).
  • the species E. coli is a particular example. Specific examples of E. coli include E. coli W3110 (ATCC 27325) and E.
  • coli MG1655 (ATCC 47076), which are derived from the prototype wild-type strain, E. coli K-12 strain. These strains are available from, for example, the American Type Culture Collection (P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to www.atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.
  • Examples of the Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes.
  • Examples of the Pantoea bacteria include Pantoea ananatis. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA.
  • a bacterium belonging to any of the genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae.
  • Pantoea ananatis strain When a Pantoea ananatis strain is bred by genetic engineering techniques, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA as described above.
  • a bacterium belonging to the family Enterobacteriaceae and modified to overexpress the yajL gene, which is able to produce either an aromatic or a non-aromatic L-amino acid, can be used.
  • the bacterium may inherently have the L-amino acid-producing ability or may be modified to have an L-amino acid-producing ability by using a mutation method or DNA recombination techniques.
  • the bacterium can be obtained by overexpressing the yajL gene in a bacterium which inherently has the ability to produce an L-amino acid.
  • the bacterium can be obtained by imparting the ability to produce an L-amino acid to a bacterium already having the yajL gene overexpressed.
  • the bacterium can produced an L-amino acid either alone or as a mixture of two or more kinds of L-amino acids
  • Examples of parental strains which can be used to derive L-arginine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strain 237 (VKPM B-7925) ( U.S. Patent Application 2002/058315 A1 ) and its derivative strains harboring mutant N-acetylglutamate synthase ( RU2215783 ), E. coli strain 382 (VKPM B-7926) ( EP1170358 A1 ), and an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein ( EP1170361 A1 ).
  • Examples of parental strains which can be used to derive L-arginine-producing bacteria also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced.
  • Examples of such genes include genes encoding N-acetyl- ⁇ -glutamylphosphate reductase ( argC ) , ornithine acetyltransferase ( argJ ), N-acetylglutamate kinase ( argB ), N-acetylomithine aminotransferase ( argD ), ornithine carbamoyltransferase ( argF ), argininosuccinate synthase ( argG ), argininosuccinate lyase ( argH ), and carbamoyl phosphate synthetase ( carAB ) .
  • parental strains which can be used to derive L-citrulline-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli mutant N -acetylglutamate synthase strains 237/pMADS11, 237/pMADS12, and 237/pMADS13 (Russian patent No. 2215783 , European patent No. 1170361 B1 , U.S. Patent No. 6790647 B2 ), E.
  • L-citrulline is an intermediate of L-arginine biosynthetic pathway
  • parent strains which can be used to derive L-citrulline-producing bacteria, include strains, in which expression of one or more genes encoding an L-arginine biosynthetic enzyme is enhanced.
  • genes include, but are not limited to, genes encoding N-acetylglutamate synthase ( argA ), N-acetylglutamate kinase ( argB ), N-acetylglutamyl phosphate reductase ( argC ), acetylomithine transaminase ( argD ), acetylomithine deacetylase ( argE ), ornithine carbamoyltransferase ( argF / I ), and carbamoyl phosphate synthetase ( carAB ), or combinations thereof.
  • argA N-acetylglutamate synthase
  • argB N-acetylglutamate kinase
  • argC N-acetylglutamyl phosphate reductase
  • argD acetylomithine transaminase
  • argE acetylomithine deacet
  • L-citrulline-producing bacterium can be also easily obtained from any L-arginine-producing bacterium, for example E. coli 382 stain (VKPM B-7926), by inactivation of argininosuccinate synthase encoded by argG gene.
  • Examples of parental strains which can be used to derive L-cysteine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli JM15 which is transformed with different cysE alleles encoding feedback-resistant serine acetyltransferases ( U.S. Patent No. 6,218,168 , Russian patent No. 2279477 ), E. coli W3110 having overexpressed genes which encode proteins suitable for secreting substances toxic for cells ( U.S. Patent No. 5,972,663 ), E. coli strains having lowered cysteine desulfohydrase activity ( JP11155571 A2 ), and E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene ( WO0127307 A1 ).
  • Examples of parental strains which can be used to derive L-glutamic acid-producing bacteria include, but are not limited to strains belonging to the genus Escherichia such as E. coli VL334thrC + ( EP 1172433 ).
  • the E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in thrC and ilvA genes ( U.S. Patent No. 4,278,765 ).
  • a wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage P1 grown on the wild-type E. coli strain K12 (VKPM B-7) cells.
  • an L-isoleucine auxotrophic strain VL334thrC (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.
  • Examples of parental strains which can be used to derive the L-glutamic acid-producing bacteria include, but are not limited to strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced.
  • Examples of such genes include genes encoding glutamate dehydrogenase ( gdhA ), glutamine synthetase ( glnA ), glutamate synthetase ( gltAB ), isocitrate dehydrogenase ( icdA ), aconitate hydratase ( acnA, acnB ), citrate synthase ( gltA ), phosphoenolpyruvate carboxylase ( ppc ), pyruvate carboxylase ( pyc ), pyruvate dehydrogenase ( aceEF, lpdA ), pyruvate kinase ( pykA, pykF ),
  • strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989 A2 , EP955368 A2 , and EP952221 A2 .
  • parental strains which can be used to derive the L-glutamic acid-producing bacteria also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L-glutamic acid biosynthesis pathway.
  • Such enzymes include isocitrate lyase ( aceA ), ⁇ -ketoglutarate dehydrogenase ( sucA ), phosphotransacetylase ( pta ), acetate kinase ( ack ), acetohydroxy acid synthase ( ilvG ), acetolactate synthase ( ilvI ), formate acetyltransferase ( pfl ), lactate dehydrogenase ( ldh ), and glutamate decarboxylase ( gadAB ) .
  • aceA isocitrate lyase
  • sucA sucA
  • pta phosphotransacetylase
  • ack acetate kinase
  • ilvG acetohydroxy acid synthase
  • ilvI acetolactate synthase
  • formate acetyltransferase pfl
  • lactate dehydrogenase ldh
  • L-glutamic acid-producing bacterium examples include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the ⁇ -ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807) ( U.S. Patent No. 5,908,768 ), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability ( U.S. Patent No. 5,393,671 ), and AJ13138 (FERM BP-5565) ( U.S. Patent No. 6,110,714 ).
  • L-glutamic acid-producing bacteria examples include mutant strains belonging to the genus Pantoea which are deficient in the ⁇ -ketoglutarate dehydrogenase activity or have a decreased ⁇ -ketoglutarate dehydrogenase activity, and can be obtained as described above.
  • Such strains include Pantoea ananatis AJ13356. ( U.S. Patent No. 6,331,419 ).
  • Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently,Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on February 19, 1998 under an accession number of FERM P-16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615.
  • Pantoea ananatis AJ13356 is deficient in ⁇ -ketoglutarate dehydrogenase activity as a result of disruption of the ⁇ KGDH-E1 subunit gene ( sucA ).
  • the above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356.
  • Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA.
  • AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.
  • Examples of parental strains which can be used to derive L-histidine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strain 24 (VKPM B-5945, RU2003677 ), E. coli strain 80 (VKPM B-7270, RU2119536 ), E. coli NRRL B-12116 - B12121 ( U.S. Patent No. 4,388,405 ), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) ( U.S. Patent No. 6,344,347 ), E . coli H-9341 (FERM BP-6674) ( EP1085087 ), and E. coli AI80/pFM201 ( U.S. Patent No. 6,258,554 ).
  • E. coli strain 24 VKPM B-5945, RU2003677
  • E. coli strain 80 VKPM B-7270, RU2119536
  • Examples of parental strains which can be used to derive L-histidine-producing bacteria also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced.
  • Examples of such genes include genes encoding ATP phosphoribosyltransferase ( hisG ), phosphoribosyl-AMP cyclohydrolase ( hisI ), phosphoribosyl-AMP cyclohydrolase/phosphoribosyl-ATP pyrophosphatase ( hisIE ), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase ( hisA ), amidotransferase ( hisH ), histidinol phosphate aminotransferase ( hisC ), histidinol phosphatase ( hisB ), and histidinol dehydrogenase ( hisD ) .
  • L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase ( Russian Patent Nos. 2003677 and 2119536 ).
  • strains having an L-histidine-producing ability include E . coli FERM-P 5038 and 5048 which have been transformed with a vector carrying a DNA encoding an L-histidine-bio synthetic enzyme ( JP 56-005099 A ), E. coli strains transformed with rht, a gene for an amino acid-export ( EP1016710 A ), E. coli 80 strain imparted with sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, RU2119536 ).
  • parental strains which can be used to derive L-isoleucine-producing bacteria include, but are not limited to, mutants having resistance to 6-dimethylaminopurine ( JP 5-304969 A ), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate ( JP 5-130882 A ).
  • recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis can also be used as parental strains ( JP 2-458 A , EP0356739 A1 , and U.S. Patent No. 5,998,178 ).
  • Examples of parental strains which can be used to derive L-leucine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121 )) or leucine analogs including ⁇ -2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine ( JP 62-34397 B and JP 8-70879 A ); E. coli strains obtained by the gene engineering method described in WO96/06926 ; and E. coli H-9068 ( JP 8-70879 A ).
  • E. coli strains resistant to leucine for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121 )
  • leucine analogs including ⁇ -2-thienylalanine, 3-hydroxyleucine,
  • the bacterium can be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which can be represented by a mutant leuA gene encoding isopropylmalate synthase freed from feedback inhibition by L-leucine ( U.S. Patent No. 6,403,342 ).
  • the bacterium can be improved by enhancing the expression of one or more genes encoding proteins which excrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes ( ygaZH genes) ( EP1239041 A2 ).
  • L-lysine-producing bacteria belonging to the family Escherichia include mutants having resistance to an L-lysine analogue.
  • the L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine is present in the medium.
  • Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), ⁇ -methyllysine, and ⁇ -chlorocaprolactam.
  • Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment.
  • bacterial strains useful for producing L-lysine include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170 ) and E. coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.
  • Examples of parental strains which can be used to derive L-lysine-producing bacteria also include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced.
  • Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase ( dapA ), aspartokinase ( lysC ), dihydrodipicolinate reductase ( dapB ), diaminopimelate decarboxylase ( lysA ), diaminopimelate dehydrogenase ( ddh ) ( U.S. Patent No.
  • the parental strains may have an increased level of expression of the gene involved in energy efficiency ( cyo ) ( EP1170376 A1 ), the gene encoding nicotinamide nucleotide transhydrogenase ( pntAB ) ( U.S. Patent No. 5,830,716 A ), the ybjE gene ( WO2005/073390 ), or combinations thereof.
  • L-Amino acid-producing bacteria may have reduced or no activity of an enzyme that catalyzes a reaction which causes a branching off from the L-amino acid biosynthesis pathway and results in the production of another compound. Also, the bacteria may have reduced or no activity of an enzyme that negatively acts on L-amino acid synthesis or accumulation.
  • enzymes involved in L-lysine production include homoserine dehydrogenase, lysine decarboxylase ( cadA, ldcC ), malic enzyme, and so forth, and strains in which activities of these enzymes are decreased or deleted are disclosed in WO95/23864 , WO96/17930 , and WO2005/010175 .
  • Expression of both the cadA and ldcC genes encoding lysine decarboxylase can be decreased in order to decrease or delete the lysine decarboxylase activity. Expression of the both genes can be decreased by, for example, the method described in WO2006/078039 .
  • L-lysine-producing bacteria can include the E. coli WC196 ⁇ cadA ⁇ ldcC/pCABD2 strain ( WO2006/078039 ).
  • the strain was constructed by introducing the plasmid pCABD2 containing lysine biosynthesis genes ( U.S. Patent No. 6,040,160 ) into the WC196 strain having disrupted cadA and ldcC genes which encode lysine decarboxylase.
  • the WC196 strain was bred from the W3110 strain, which was derived from E . coli K-12 by replacing the wild-type lysC gene on the chromosome of the W3110 strain with a mutant lysC gene encoding a mutant aspartokinase III in which threonine at position 352 was replaced with isoleucine, resulting in desensitization of the feedback inhibition by L-lysine ( U.S. Patent No. 5,661,012 ), and conferring AEC resistance to the resulting strain ( U.S. Patent No. 5,827,698 ).
  • the WC196 strain was designated E.
  • the WC 196 ⁇ cadA ⁇ ldcC strain itself is also an exemplary L-lysine-producing bacterium.
  • the WC196 ⁇ cadA ⁇ ldcC was designated AJ110692 and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on October 7, 2008 as an international deposit under an accession number of FERM BP-11027.
  • L-methionine-producing bacteria and parent strains which can be used to derive L-methionine-producing bacteria include, but are not limited to, Escherichia bacteria strains such as strains AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRL B-12402) (patent GB2075055 ); strains 218 (VKPM B-8125) (patent RU2209248 ) and 73 (VKPM B-8126) (patent RU2215782 ) resistant to norleucine, the L-methionine analog, or the like.
  • Escherichia bacteria strains such as strains AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRL B-12402) (patent GB2075055 ); strains 218 (VKPM B-8125) (patent
  • coli 73 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on May 14, 2001 under accession number VKPM B-8126, and was converted to an international deposit under the Budapest Treaty on February 1, 2002. Furthermore, a methionine repressor-deficient strain and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis such as homoserine transsuccinylase and cystathionine ⁇ -synthase ( JP 2000-139471 A ) can also be used as parent strains.
  • VKPM National Collection of Industrial Microorganisms
  • L-ornithine-producing bacterium can be easily obtained from any L-arginine-producing bacterium, for example E. coli 382 stain (VKPM B-7926), by inactivation of ornithine carbamoyltransferase encoded by both argF and argI genes. Methods for inactivation of ornithine carbamoyltransferase are described herein.
  • Examples of parental strains which can be used to derive L-phenylalanine-producing bacteria include, but are limited to strains belonging to the genus Escherichia such as E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197), E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene ( U.S. Patent No. 5,354,672 ), E. coli MWEC101-b ( KR8903681 ), E . coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 ( U.S. Patent No. 4,407,952 ). Also, E.
  • coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662), and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used as a parental strain ( EP488424 B1 ).
  • L-phenylalanine-producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used ( U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1 ).
  • Examples of parental strains which can be used to derive L-proline-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli 702ilvA (VKPM B-8012) which is deficient in the ilvA gene and is able to produce L-proline ( EP1172433 A1 ).
  • the bacterium can be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis.
  • Examples of genes which can be used in L-proline-producing bacteria include the proB gene encoding glutamate kinase with desensitized feedback inhibition by L-proline ( DE3127361 A1 ).
  • the bacterium can be improved by enhancing the expression of one or more genes encoding proteins excreting L-amino acid from bacterial cell.
  • genes are exemplified by b2682 and b2683 genes ( ygaZH genes) ( EP1239041 A2 ).
  • Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 ( GB Patent 2075056 ), VKPM B-8012 ( Russian patent application No. 2000124295 ), plasmid mutants described in DE3127361 A1 , and plasmid mutants described by Bloom F.R. et al. in "The 15th Miami winter symposium", 1983, p.34 .
  • Examples of parental strains which can be used to derive L-threonine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli TDH-6/pVIC40 (VKPM B-3996) ( U.S. Patent No. 5, 175, 107 , U.S. Patent No. 5,705,371 ), E. coli 472T23/pYN7 (ATCC 98081) ( U.S. Patent No.5,631,157 ), E. coli NRRL-21593 ( U.S. Patent No. 5,939,307 ), E. coli FERM BP-3756 ( U.S. Patent No. 5,474,918 ), E.
  • E. coli TDH-6/pVIC40 VKPM B-3996
  • U.S. Patent No. 5, 175, 107 U.S. Patent No. 5,705,371
  • E. coli 472T23/pYN7 ATCC 98081
  • the strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine.
  • the strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA * BC operon which includes a mutant thrA gene into a RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine.
  • the strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics (Russian Federation, 117105 Moscow, Nagatinskaya Street 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on April 7, 1987 under the accession number VKPM B-3996.
  • VKPM Russian National Collection of Industrial Microorganisms
  • E. coli VKPM B-5318 ( EP0593792 A1 ) may also be used as a parental strain for deriving L-threonine-producing bacteria.
  • the strain B-5318 is prototrophic with regard to isoleucine; and a temperature-sensitive lambda-phage C1 repressor and PR promoter replace the regulatory region of the threonine operon in plasmid pVIC40.
  • the strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on May 3, 1990 under the accession number of VKPM B-5318.
  • the bacterium can be additionally modified to enhance expression of one or more of the following genes:
  • the thrA gene which encodes aspartokinase I and homoserine dehydrogenase I of E. coli has been elucidated ( KEGG, Kyoto Encyclopedia of Genes and Genomes , entry No. b0002; GenBank accession No. NC_000913.2; nucleotide positions: 337 to 2,799; Gene ID: 945803).
  • the thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12.
  • the thrB gene which encodes homoserine kinase of E. coli has been elucidated (KEGG entry No. b0003; GenBank accession No. NC_000913.2; nucleotide positions: 2,801 to 3,733; Gene ID: 947498).
  • the thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12.
  • the thrC gene which encodes threonine synthase of E. coli has been elucidated (KEGG entry No. b0004; GenBank accession No. NC_000913.2; nucleotide positions: 3,734 to 5,020; Gene ID: 945198).
  • the thrC gene is located between the thrB and yaaX genes on the chromosome of E. coli K-12. All three genes function as a single threonine operon thrABC.
  • the attenuator region which affects the transcription is desirably removed from the operon ( WO2005049808 A1 , WO2003097839 A1 ).
  • mutant thrA gene which encodes aspartokinase I and homoserine dehydrogenase I resistant to feedback inhibition by L-threonine, as well as, the thrB and thrC genes can be obtained as one operon from the well-known plasmid pVIC40 which is present in the L-threonine-producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371 .
  • the rhtA gene which encodes a protein of the threonine and homoserine efflux system (an inner membrane transporter) of E. coli has been elucidated (KEGG entry No. b0813; GenBank accession No. NC_000913.2; nucleotide positions: 848,433 to 849,320, complement; Gene ID: 947045).
  • the rhtA gene is located between the dps and ompX genes on the chromosome of E. coli K-12 close to the glnHPQ operon, which encodes components of the glutamine transport system.
  • the rhtA gene is identical to the ybiF gene (KEGG entry No. B0813).
  • the asd gene which encodes aspartate-p-semialdehyde dehydrogenase of E. coli has been elucidated (KEGG entry No. b3433; GenBank accession No. NC_000913.2; nucleotide positions: 3,571,798 to 3,572,901, complement; Gene ID: 947939).
  • the asd gene is located between the glgB and gntU gene on the same strand ( yhgN gene on the opposite strand) on the chromosome of E. coli K-12.
  • the aspC gene which encodes aspartate aminotransferase of E. coli has been elucidated (KEGG entry No. b0928; GenBank accession No. NC_000913.2; nucleotide positions: 983,742 to 984,932, complement; Gene ID: 945553).
  • the aspC gene is located between the ycbL gene on the opposite strand and the ompF gene on the same strand on the chromosome of E. coli K-12.
  • parental strains which can be used to derive the L-tryptophan-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene ( U.S. Patent No. 5,756,345 ), E.
  • coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan ( U.S. Patent No. 6,180,373 ), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase ( U.S. Patent No. 4,371,614 ), and E.
  • coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced ( WO9708333 , U.S. Patent No. 6,319,696 ) may be used.
  • L-tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene may also be used ( U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1 ).
  • Examples of parental strains which can be used to derive the L-tryptophan-producing bacteria also include strains in which one or more activities of the enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced.
  • the anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes.
  • Specific examples of strains having such a mutation include an E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 ( WO 94/08031 ), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
  • Examples of parental strains which can be used to derive the L-tryptophan-producing bacteria also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced ( JP 57-71397 A , JP 62-244382 A , U.S. Patent No. 4,371,614 ).
  • L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons ( trpBA ) .
  • the tryptophan synthase consists of ⁇ and ⁇ subunits which are encoded by the trpA and trpB genes, respectively.
  • L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon ( WO2005/103275 ).
  • parental strains which can be used to derive L-valine-producing bacteria include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon ( U.S. Patent No. 5,998,178 ). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the L-valine that is produced. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.
  • Examples of parental strains for deriving L-valine-producing bacteria also include mutants having a mutation of aminoacyl-tRNA synthetase ( U.S. Patent No. 5,658,766 ).
  • E. coli VL1970 which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used.
  • E. coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on June 24, 1988 under the accession number VKPM B-4411.
  • mutants requiring lipoic acid for growth and/or lacking H + -ATPase can also be used as parental strains ( WO96/06926 ).
  • Parent strains for deriving L-valine-producing bacteria also include E. coli H81 strain (VKPM B-8066; see, for example, EP1942183 B1 ), NRRL B-12287 and NRRL B-12288 ( U.S. Patent No. 4,391,907 ), VKPM B-4411 ( U.S. Patent No. 5,658,766 ), and VKPM B-7707 (European patent application EP1016710 A2 ).
  • the bacterium used in the present invention belonging to the family Enterobacteriaceae has been modified to overexpress the yajL gene.
  • a bacterium modified to overexpress the yajL gene can mean that the bacterium has been modified in such a way that in the modified bacterium the total enzymatic activity of the corresponding gene protein product such as YajL is increased as compared with, or the expression level of the yajL gene is higher than that level in, a non-modified strain, for example, a wild-type or parental strain as described above and hereinafter.
  • a non-modified strain serving as a reference for the above comparison can include a wild-type strain of a microorganism belonging to the genus Escherichia such as the E. coli MG1655 strain (ATCC 47076), and W3110 strain (ATCC 27325).
  • the phrase "the yajL gene is overexpressed" can mean that the total enzymatic activity of the corresponding gene protein product such as the YajL protein is increased by, for example, introducing and/or increasing the copy number of the yajL gene in bacterial genome, or increasing the activity per molecule (may be referred to as a specific activity) of the protein encoded by said gene, as compared with a non-modified strain.
  • the bacterium can be modified so that the activity of the YajL protein per cell is increased to 150% or more, 200% or more, 300% or more, of the activity of a non-modified strain.
  • the chaperone activity of YajL can be used to determine a specific enzymatic activity of the protein per mg.
  • citrate synthase can be used as a control protein to measure refolding activity of YajL under reducing or oxidizing conditions ( Kthiri F. et al., J. Biol. Chem., 2010, 285:10328-10336 ).
  • the protein concentration can be determined by the Bradford protein assay ( Bradford M.M., Anal. Biochem., 1976, 72:248-254 ) using bovine serum albumin as a standard.
  • the phrase "the yajL gene is overexpressed” can also mean that the expression level of the yajL gene is higher than that level in a non-modified strain. Therefore, the phrase “the yajL gene is overexpressed” is equivalent to the phrase “expression of the yajL gene is enhanced”.
  • Methods which can be used to enhance expression of the yajL gene include, but are not limited to, increasing the yajL gene copy number in bacterial genome (in the chromosome and/or in the autonomously replicable plasmid) and/or introducing the yajL gene into a vector that is able to increase the copy number and/or the expression level of the yajL gene in a bacterium of the family Enterobacteriaceae according to genetic engineering methods known to the one skilled in the art.
  • vectors include, but are not limited to, broad-host-range plasmids such as pMW1 18/119, pBR322, and pUC19.
  • Multiple copies of the yajL gene can also be introduced into the chromosomal DNA of a bacterium by, for example, homologous recombination or Mu-driven integration. Homologous recombination can be carried out using sequence with multiple copies in the chromosomal DNA. Sequences with multiple copies in the chromosomal DNA include, but are not limited to, repetitive DNA or inverted repeats present at the end of a transposable element.
  • yajL gene into a transposon and allow it to be transferred to introduce multiple copies of the yajL gene into the chromosomal DNA.
  • Mu-driven integration more than 3 copies of the gene can be introduced into the chromosomal DNA during a single act ( Akhverdyan V.Z. et al., Biotechnol. ( Russian), 2007, 3:3-20 ).
  • Enhancing of the yajL gene expression can also be achieved by increasing the expression level of the yajL gene by modification of adjacent regulatory regions of the yajL gene or introducing native and/or modified foreign regulatory regions.
  • Regulatory regions or sequences can be exemplified by promoters, enhancers, attenuators and termination signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements (e.g., regions to which repressors or inducers bind and/or binding sites for transcriptional and translational regulatory proteins, for example, in the transcribed mRNA).
  • RBS ribosome-binding sites
  • the exemplary promoters enhancing the yajL gene expression can be the potent promoters that are stronger than the native yajL promoter.
  • the lac promoter, the trp promoter, the trc promoter, the tac promoter, the P R or the P L promoters of lambda phage are all known to be potent promoters.
  • Potent promoters providing a high level of gene expression in a bacterium belonging to the family Enterobacteriaceae can be used.
  • the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of the yajL gene to obtain a stronger promoter function, thus resulting in the increased transcription level of the yajL gene located downstream of the promoter.
  • substitution of several nucleotides in the Shine-Dalgarno (SD) sequence, and/or in the spacer between the SD sequence and the start codon, and/or a sequence immediately upstream and/or downstream from the start codon in the ribosome-binding site greatly affects the translation efficiency of mRNA. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon ( Gold L. et al., Annu. Rev. Microbiol., 1981, 35:365-403 ; Hui A. et al., EMBO J., 1984, 3:623-629 ).
  • the copy number, presence or absence of the gene can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, and fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • the level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting and quantitative RT-PCR.
  • the amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), or mass spectrometry analysis of the protein samples.
  • the yajL gene encodes the oxidative-stress-resistance chaperone YajL ( KEGG, Kyoto Encyclopedia of Genes and Genomes , entry No. b0424; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. Q46948).
  • the yajL gene (GenBank accession No. NC_000913.2; nucleotide positions: 442275 to 442865, complement; Gene ID: 945066) is located between the panE gene on the same strand and the thiI gene on the opposite strand on the chromosome of E. coli strain K-12.
  • the nucleotide sequence of the yajL gene and the amino acid sequence of the YajL protein encoded by the yajL gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • This phenotype is now attributed to the adjacent thiI gene (T. Begley, personal communication cited in Mueller E.G. et al., Identification of a gene involved in the generation of 4-thiouridine in tRNA, Nucleic Acids Res., 1998, 26(11):2606-2610 ).
  • the yajL gene is not limited to the gene shown in SEQ ID NO: 1, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NO: 1, and which encode variants of the YajL protein.
  • a variant protein can mean a protein which has one or several changes in the sequence compared with SEQ ID NO: 2, whether they are substitutions, deletions, insertions, and/or additions of one or several amino acid residues, but still maintains an activity or function similar to that of the YajL protein, or the three-dimensional structure of the YajL protein is not significantly changed relative to the wild-type or non-modified protein.
  • the number of changes in the variant protein depends on the position in the three-dimensional structure of the protein or the type of amino acid residues. It can be, but is not strictly limited to, 1 to 30, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2.
  • the protein variants encoded by the yajL gene may have a homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 98%, or not less than 99% with respect to the entire amino acid sequence shown in SEQ ID NO: 2, as long as the activity or function of the YajL protein is maintained, or the three-dimensional structure of YajL is not significantly changed relative to the wild-type or non-modified protein.
  • the exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s).
  • the representative conservative mutation is a conservative substitution.
  • the conservative substitution can be, but is not limited to, a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gin and Asn, if the substitution site is a polar amino acid; among Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having hydroxyl group.
  • conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution Asn, Glu, Lys, His, Asp or Arg for Gln, substitution Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe
  • the exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation(s) provided that the mutation(s) is/are compensated by one or more secondary mutation(s) in the different position(s) of amino acids sequence so that the activity or function of the variant protein is maintained and similar to that of the YajL protein, or the three-dimensional structure of YajL is not significantly changed relative to the wild-type or non-modified protein.
  • BLAST search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci.
  • the computer program BLAST calculates three parameters: score, identity and similarity.
  • the FASTA search method is described by Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods Enzymol., 1990, 183:63-98 ).
  • the ClustalW method is described by Thompson J.D. et al. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res., 1994, 22:4673-4680 ).
  • the yajL gene can be a variant nucleotide sequence.
  • the phrase "a variant nucleotide sequence” can mean a nucleotide sequence which encodes "a variant protein” using any synonymous amino acid codons according to the standard genetic code table (see, e.g., Lewin B., "Genes VIII", 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458 ). Therefore, the yajL gene can be a variant nucleotide sequence due to degeneracy of genetic code.
  • a variant nucleotide sequence can also mean, but is not limited to, a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1, or a probe which can be prepared from the nucleotide sequence under stringent conditions provided that it encodes active or functional protein.
  • “Stringent conditions” include those under which a specific hybrid, for example, a hybrid having homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 60%, not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed.
  • a specific hybrid for example, a hybrid having homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 60%, not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed.
  • stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt concentration of 1 ⁇ SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulphate), or in another example, 0.1 ⁇ SSC, 0.1% SDS at 60°C or 65°C.
  • Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham HybondTM-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes.
  • the washing step can be performed 2 to 3 times.
  • a part of the sequence complementary to the sequences shown in SEQ ID NO: 1 may also be used.
  • Such a probe can be produced by PCR using oligonucleotides as primers prepared on the basis of the sequence shown in SEQ ID NO: 1 and a DNA fragment containing the nucleotide sequence as a template.
  • the length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
  • the washing conditions after hybridization can be exemplified by 2 ⁇ SSC, 0.1% SDS at 50°C, 60°C or 65°C.
  • the yajL gene encoding the YajL protein, and the variant nucleotide sequences encoding variant proteins of YajL protein can be obtained by PCR (polymerase chain reaction; refer to White T.J.
  • a wild-type protein can mean a native protein naturally produced by a wild-type or parent bacterial strain of the family Enterobacteriaceae, for example, by the wild-type E. coli MG1655 strain.
  • a wild-type protein can be encoded by the "wild-type gene", or the "non-modified gene” naturally occurring in genome of a wild-type bacterium.
  • the bacterium as described herein can be obtained by modifying a bacterium inherently having an ability to produce an L-amino acid to overexpress the yajL gene, for example, by introducing the aforementioned DNAs into the bacterium.
  • the bacterium as described herein can be obtained by imparting the ability to produce an L-amino acid to a bacterium already modified to overexpress the yajL gene.
  • the bacterium can have, in addition to the properties already mentioned, other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence.
  • a method of the present invention includes the method for producing an L-amino acid such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, or a mixture thereof.
  • L-amino acid such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine
  • the method for producing an L-amino acid can include the steps of cultivating the bacterium in a culture medium to allow the L-amino acid to be produced, excreted, and/or accumulated in the culture medium or in the bacterial cells, and collecting the L-amino acid from the culture medium and/or the bacterial cells.
  • the L-amino acid can be produced in a salt or a hydrate form thereof, or a combination thereof.
  • sodium, potassium, and ammonium, salts of the L-amino acid can be produced by the method.
  • the L-amino acid can be produced in an adduct form thereof with, for example, another organic or inorganic compound.
  • a monochlorhydrate salt of an L-amino acid can be produced by the method such as monochlorhydrate salt of L-lysine.
  • the cultivation of the bacterium, and collection and purification of L-amino acid from the medium may be performed in a manner similar to conventional fermentation methods wherein L-amino acid is produced using a microorganism.
  • the culture medium for production of the L-amino acid can be either a synthetic or natural medium such as a typical medium that contains a carbon source, a nitrogen source, a sulphur source, inorganic ions, and other organic and inorganic components as required.
  • saccharides such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as ethanol, glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and fatty acid can be used.
  • saccharides such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches
  • alcohols such as ethanol, glycerol, mannitol, and sorbitol
  • organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid
  • fatty acid can be used.
  • inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soy bean hydrolyzates; ammonia gas; and aqueous ammonia
  • the sulphur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, and manganese sulphate.
  • Vitamins such as vitamin B1, required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, or yeast extract may be present in appropriate, even if trace, amounts. Other than these, small amounts of calcium phosphate, iron ions, and manganese ions may be added, if necessary.
  • Cultivation can be performed under aerobic conditions for 16 to 72 h, or for 32 to 68 h; the culture temperature during cultivation can be controlled within 30 to 45°C, or within 30 to 37°C; and the pH can be adjusted between 5 and 8, or between 6 and 7.5.
  • the pH can be adjusted by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.
  • solids such as cells and cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid can be recovered from the fermentation liquor by any combination of conventional techniques such as concentration, ion-exchange chromatography, and crystallization.
  • Example 1 Construction of the E. coli L-valine-producing strain modified to overexpress the yajL gene
  • the chromosome of the chloramphenicol-resistant E. coli strain MG1655 which contains a tac -like promoter having the structure of the -35 region as TGGCAA (from 5'-end to 3'-end) upstream lacZ (refer to Table 1, clone 7 in Katashkina J.L. et al., Tuning the expression level of a gene located on a bacterial chromosome, Mol. Biol. (Mosk., in Russian), 2005, 39(5):719-726 ) was used as the template in PCR reaction.
  • DNA-fragment 1 (1,768 bp) (SEQ ID NO: 5) was purified in an agarose gel and used for electroporation of the strain E. coli MG1655 (ATCC 47076) containing the plasmid pKD46 with a temperature-sensitive replication origin.
  • the plasmid pKD46 Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci.
  • Electrocompetent cells were prepared as follows: E. coli MG1655 cells were grown overnight at 30°C in LB-medium ( Sambrook, J. and Russell, D.W. "Molecular Cloning: A Laboratory Manual”, 3rd ed., Cold Spring Harbor Laboratory Press (2001 )) containing ampicillin (100 mg/L), and the culture was diluted 100 times with 5 mL of SOB-medium ( Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press (1989 )) containing ampicillin (100 mg/L) and L-arabinose (1 mM).
  • the obtained culture was grown with aeration (250 rpm) at 30°C to an OD 600 of ⁇ 0.6 and then made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H 2 O. Electroporation was performed using 70 ⁇ L of cells and ⁇ 100 ng of the DNA-fragment 1. Then, cells were incubated with 1 mL of SOC-medium ( Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press (1989 )) at 37°C for 2.5 h, placed onto the plates containing LB-medium ( Sambrook, J. and Russell, D.W.
  • Mutants containing the replacement of a promoter region of the yajL gene marked with Cm R -gene ( cat ) were verified by PCR using locus-specific primers P3 (SEQ ID NO: 6) and P4 (SEQ ID NO: 7). Conditions for PCR were as follows: denaturation for 3 min at 94°C; profile for 30 cycles: 30 sec at 94°C, 30 sec at 58°C, 2 min at 72°C; final elongation for 6 min at 72°C.
  • DNA-fragment 2 obtained in the reaction with the chromosomal DNA from the parent strain E. coli MG1655 as the template, was 966 bp in length (SEQ ID NO: 8).
  • DNA-fragment 3 obtained in the reaction with the chromosomal DNA from the mutant strain E. coli MG1655 P tac7 - yajL as a template, was 1,820 bp in length (SEQ ID NO: 9).
  • the yajL gene under control of P tac7 promoter was introduced into valine-producing E. coli strain H81 (Russian patent No. 2355763 , EP1942183 B1 ) by P1-transduction ( Miller J.H. "Experiments in molecular genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor (1972 )).
  • the E. coli MG1655 P tac 7 -yajL strain was used as a donor.
  • the strain H81 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on January 30, 2001 under accession number VKPM B-8066 and then converted to a deposit under the Budapest Treaty on February 1, 2002.
  • the E. coli H81 mutants harboring the P tac7 - yajL cassette were selected on the plates containing LB-medium, agar (1.5%) and chloramphenicol (20 mg/L). Thus the strain E . coli H81 P tac7 - yajL was obtained. Replacement of the promoter region of the yajL gene was verified by PCR as described in Section 1.2 of Example 1.
  • the modified E . coli H81 P tac7 -yajL and the control E. coli H81 strains were each cultivated at 32°C for 18 hours in LB-medium (also referred to as lysogenic broth or Luria-Bertani medium as described in Sambrook, J. and Russell, D.W. "Molecular Cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press (2001 )). Then, 0.2 mL of the obtained culture was inoculated into 2 mL of a fermentation medium in 20 ⁇ 200-mm test tubes and cultivated at 32°C for 66 hours on a rotary shaker at 250 rpm to an OD 550 of ⁇ 29 until glucose consumption.
  • composition of the fermentation medium was as follows: Glucose 60.0 (NH 4 ) 2 SO 4 15.0 KH 2 PO 4 1.5 MgSO 4 ⁇ 7H 2 O 1.0 Thiamine-HCl 0.1 CaCO 3 25.0 LB-medium 10% (v/v)
  • the fermentation medium was sterilized at 116°C for 30 min, except that glucose and CaCO 3 were sterilized separately as follows: glucose at 110°C for 30 min and CaCO 3 at 116°C for 30 min.
  • the pH was adjusted to 7.0 by KOH solution.
  • TLC thin-layer chromatography
  • SorbfilTM silica gel containing non-fluorescent indicator SorbpolymerTM, Krasnodar, Russian Federation.
  • Samples were applied onto the plates with the Camag LinomatTM 5 sample applicator.
  • the Sorbfil plates were developed with a mobile phase consisting of propan-2-ol : ethylacetate : 25% aqueous ammonia : water (16: 16: 5 : 10, v/v).
  • a solution of ninhydrin (2%, w/v) in acetone was used as the visualizing reagent.
  • plates were dried and scanned with the Camag TLC Scanner 3TM in absorbance mode with detection at 520 nm using winCATSTM software (version 1.4.2).
  • Example 3 Construction of the E. coli L-phenylalanine-producing strain modified to overexpress the yajL gene
  • E. coli DV269 (TyrA-LAA) strain, also known as E . coli MG1655 htrE :(P L -yddG ) [MUD aroG 4 -pheA fbr -aroL ], as described in Section 1.3 of Example 1.
  • the E. coli MG1655 P tac7 -yajL strain (see Example 1.1) was used as a donor. Construction of the E. coli DV269 (TyrA-LAA) strain is described in Doroshenko V.G.
  • 0.2 mL of the obtained culture was inoculated into 2 mL of a fermentation medium in 20 ⁇ 200-mm test tubes and cultivated at 34°C for 30 hours on a rotary shaker at 240 rpm to an OD 540 of ⁇ 6.5 - 7 until glucose consumption.
  • composition of the fermentation medium is as follows: Glucose 50.0 (NH 4 ) 2 SO 4 0.6 K 2 HPO 4 ⁇ 3H 2 O 0.6 FeSO 4 ⁇ 7H 2 O 0.01 MnSO 4 ⁇ 5H 2 O 0.01 Yeast extract 2.0 CaCO 3 20.0
  • Glucose was sterilized separately.
  • CaCO 3 was dry-heat sterilized at 180°C for 2 h.
  • TLC thin-layer chromatography
  • the DNA-fragments from the chromosome of the above-described E. coli MG1655 P tac7 - yajL strain are transferred to the arginine-producing E. coli strain 382 by PI-transduction ( Miller, J.H. (1972), Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY ) to obtain the strain 382 P tac7 -yajL.
  • the strain 382 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on April 10, 2000 under the accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
  • VKPM National Collection of Industrial Microorganisms
  • E. coli strains 382 and 382 P tac7 - yajL are separately cultivated with shaking (220 rpm) at 37°C for 18 h in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 ⁇ 200-mm test tubes and cultivated at 32°C for 48 h on a rotary shaker (220 rpm).
  • a solution of ninhydrin (2%) in acetone is used as a visualizing reagent.
  • a spot containing L-arginine is cut out, L-arginine is eluted with 0.5% water solution of CdCl 2 , and the amount of L-arginine is estimated spectrophotometrically at 540 nm.
  • composition of the fermentation medium is as follows: Glucose 48.0 (NH 4 ) 2 SO 4 35.0 KH 2 PO 4 2.0 MgSO 4 ⁇ 7H 2 O 1.0 Thiamine-HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO 3 5.0
  • Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180°C for 2 h. The pH is adjusted to 7.0.
  • the DNA fragments from the chromosome of the above-described E . coli MG1655 P tac7 - yajL strain are transferred to the L-cysteine-producing E. coli strain JM15(ydeD) by P1-transduction to obtain the strain JM15(ydeD) P tac7 -yajL.
  • E . coli JM15(ydeD) is a derivative of E. coli JM15 ( U.S. Patent No. 6,218,168 ), which is transformed with DNA having the ydeD gene encoding a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid ( U.S. Patent No. 5,972,663 ).
  • the DNA fragments from the chromosome of the above-described E . coli MG1655 P tac7 - yajL strain are transferred to the E. coli L-glutamate-producing strain VL334thrC + ( EP1172433 A1 ) by P1-transduction to obtain the strain VL334thrC + P tac7 - yajL.
  • strain VL334thrC + was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on December 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004.
  • VKPM National Collection of Industrial Microorganisms
  • E. coli strains VL334thrC + and VL334thrC + P tac7 - yajL are separately cultivated for 18-24 h at 37°C on L-agar plates. Then, one loop of the cells is transferred into test tubes containing 2 mL of fermentation medium. Cultivation is carried out at 30°C for 3 days with shaking.
  • composition of the fermentation medium is as follows: Glucose 60.0 (NH 4 ) 2 SO 4 25.0 KH 2 PO 4 2.0 MgSO 4 ⁇ 7H 2 O 1.0 Thiamine-HCl 0.1 L-isoleucine 0.07 CaCO 3 25.0
  • Glucose and CaCO 3 are sterilized separately. The pH is adjusted to 7.2.
  • the DNA fragments from the chromosome of the above-described E. coli MG1655 P tac7 -yajL strain are transferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121 ) by P1-transduction to obtain the strain 57 P tac7 -yajL.
  • the strain 57 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on May 19, 1997 under the accession number VKPM B-7386.
  • E. coli strains 57 and 57 P tac7 - yajL are separately cultivated for 18-24 h at 37°C on L-agar plates.
  • the strains are grown on a rotary shaker (250 rpm) at 32°C for 18 h in 20 ⁇ 200-mm test tubes containing 2 mL of L-broth ( Sambrook, J. and Russell, D.W. (2001) "Molecular Cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press ) supplemented with sucrose (4%).
  • the fermentation medium is inoculated with 0.2 mL of seed material (10%).
  • the fermentation is performed in 2 mL of a minimal fermentation medium in 20 ⁇ 200-mm test tubes.
  • composition of the fermentation medium is as follows: Glucose 60.0 (NH 4 ) 2 SO 4 25.0 K 2 HPO 4 2.0 MgSO 4 ⁇ 7H 2 O 1.0 Thiamine-HCl 0.01 CaCO 3 25.0
  • Glucose is sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180°C for 2 h. The pH is adjusted to 7.2.
  • the DNA fragments from the chromosome of the above-described E. coli MG1655 P tac7 - yajL strain are transferred to the L-lysine-producing E. coli strain AJ11442 by P1-transduction to obtain the AJ11442 P tac7 - yajL strain.
  • the strain AJ11442 was deposited in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (currently Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on May 1, 1981 under a deposition number of FERM P-5084. Then it was converted to an international deposit under the provisions of the Budapest Treaty on October 29, 1987 and received an accession number of FERM BP-1543.
  • the pCABD2 plasmid includes the dapA gene encoding dihydrodipicolinate synthase having a mutation which desensitizes feedback inhibition by L-lysine, the lysC gene encoding aspartokinase III having a mutation which desensitizes feedback inhibition by L-lysine, the dapB gene encoding dihydrodipicolinate reductase, and the ddh gene encoding diaminopimelate dehydrogenase ( U.S. Patent No. 6,040,160 ).
  • E. coli strains AJ11442 and AJ11442 P tac7 - yajL are separately cultivated in L-medium containing streptomycin (20 mg/L) at 37°C, and 0.3 mL of the obtained culture is inoculated into 20 mL of the fermentation medium containing the required drugs in a 500-mL flask.
  • the cultivation is carried out at 37°C for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm.
  • the amounts of L-lysine and residual glucose in the medium are determined by a known method (Biotech-analyzerTM AS210, Sakura Seiki Co.). Then, the yield of L-lysine is calculated relative to consumed glucose for each of the strains.
  • composition of the fermentation medium is as follows: Glucose 40.0 (NH 4 ) 2 SO 4 24.0 K 2 HPO 4 1.0 MgSO 4 ⁇ 7H 2 O 1.0 FeSO 4 ⁇ 7H 2 O 0.01 MnSO 4 ⁇ 5H 2 O 0.01 Yeast extract 2.0
  • the pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180°C for 2 h and added to the medium for a final concentration of 30 g/L.
  • the DNA fragments from the chromosome of the above-described E. coli MG1655 P tac7 - yajL strain are transferred to the proline-producing E . coli strain 702ilvA by P1-transduction to obtain the strain 702ilvA P tac7 -yajL.
  • the strain 702ilvA was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on July 18, 2000 under the accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
  • E. coli strains 702ilvA and 702ilvA P tac7 - yajL are separately cultivated for 18-24 h at 37°C on L-agar plates. Then, these strains are cultivated under the same conditions as in Example 7 (Production of L-glutamic acid).
  • the DNA fragments from the chromosome of the above-described E. coli MG1655 P tac7 - yajL strain are transferred to the L-threonine-producing E. coli strain VKPM B-3996 by P1-transduction to obtain the strain B-3996 P tac7 -yajL.
  • the strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics ( Russian Federation, 117105 Moscow, Nagatinskaya Street, 3-A) under the accession number RIA 1867.
  • VKPM National Collection of Industrial Microorganisms
  • E. coli strains B-3996 and B-3996 P tac7 - yajL are separately cultivated for 18-24 h at 37°C on L-agar plates.
  • the strains are grown on a rotary shaker (250 rpm) at 32°C for 18 h in 20 ⁇ 200-mm test tubes containing 2 mL of L-broth ( Sambrook, J. and Russell, D.W. (2001) "Molecular Cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press ) supplemented with glucose (4%).
  • the fermentation medium is inoculated with 0.2 mL (10%) of seed material.
  • the fermentation is performed in 2 mL of minimal medium in 20 ⁇ 200-mm test tubes. Cells are grown for 65 h at 32°C with shaking at 250 rpm.
  • a solution of ninhydrin (2%) in acetone is used as a visualizing reagent.
  • a spot containing L-threonine is cut out, L-threonine is eluted with 0.5% water solution of CdCl 2 , and the amount of L-threonine is estimated spectrophotometrically at 540 nm.
  • composition of the fermentation medium is as follows: Glucose 80.0 (NH 4 ) 2 SO 4 22.0 NaCl 0.8 KH 2 PO 4 2.0 MgSO 4 ⁇ 7H 2 O 0.8 FeSO 4 ⁇ 7H 2 O 0.02 MnSO 4 ⁇ 5H 2 O 0.02 Thiamine-HCl 0.0002 Yeast extract 1.0 CaCO 3 30.0
  • Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is sterilized by dry-heat at 180°C for 2 h.
  • the pH is adjusted to 7.0.
  • the antibiotic is introduced into the medium after sterilization.
  • the DNA fragments from the chromosome of the above-described E. coli MG1655 P tac7 - yajL strain are transferred to the L-tryptophan-producing E. coli strain SV164(pGH5) by PI-transduction to obtain the strain SV164(pGH5) P tac7 -yajL.
  • the strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan.
  • the plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine.
  • the strain SV164(pGH5) was described in detail in U.S. patent No. 6,180,373 or EP0662143 B1 .
  • E. coli strains SV164(pGH5) and SV164(pGH5) P tac7 - yajL are separately cultivated with shaking at 37°C for 18 h in 3 mL of nutrient broth supplemented with tetracycline (20 mg/L, marker of pGH5 plasmid).
  • the obtained cultures (0.3 mL each) are inoculated into 3 mL of a fermentation medium containing tetracycline (20 mg/L) in 20 ⁇ 200-mm test tubes, and cultivated at 37°C for 48 h with a rotary shaker at 250 rpm. After cultivation, the amount of L-tryptophan which accumulates in the medium is determined by TLC.
  • the 10 ⁇ 15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing non-fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russian Federation) are used.
  • a solution of ninhydrin (2%) in acetone is used as a visualizing reagent.
  • the fermentation medium components are listed in Table 3, but should be sterilized in separate groups (A, B, C, D, E, F, and H), as shown, to avoid adverse interactions during sterilization. Table 3.
  • the DNA fragments from the chromosome of the above-described E. coli MG1655 P tac7 - yajL strain are transferred to the L-citrulline producing E . coli strain 382 ⁇ argG by P1-transduction to obtain the strain 382 ⁇ argG P tac7 -yajL.
  • the strain 382 ⁇ argG is obtained by deletion of argG gene on the chromosome of 382 strain (VKPM B-7926) by the method initially developed by Datsenko K.A. and Wanner B.L. called " ⁇ Red/ET-mediated integration" ( Datsenko K.A.
  • E. coli strains 382 ⁇ argG and 382 ⁇ argG P tac7 - yajL are separately cultivated with shaking at 37°C for 18 h in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 ⁇ 200-mm test tubes and cultivated at 32°C for 48 h on a rotary shaker.
  • a solution of ninhydrin (2%) in acetone is used as a visualizing reagent.
  • a spot containing citrulline is cut out, L-citrulline is eluted with 0.5% water solution of CdCl 2 , and the amount of L-citrulline is estimated spectrophotometrically at 540 nm.
  • composition of the fermentation medium is as follows: Glucose 48.0 (NH 4 ) 2 SO 4 35.0 KH 2 PO 4 2.0 MgSO 4 ⁇ 7H 2 O 1.0 Thiamine-HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO 3 5.0
  • Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180°C for 2 h. The pH is adjusted to 7.0.
  • the DNA fragments from the chromosome of the above-described E . coli MG1655 P tac7 - yajL strain are transferred to the L-ornithine producing E . coli strain 382 ⁇ argF ⁇ argI by P1-transduction to obtain the strain 382 ⁇ argF ⁇ argI P tac7 -yajL.
  • the strain 382 ⁇ argF ⁇ argI is obtained by consecutive deletion of argF and argI genes on the chromosome of 382 strain (VKPM B-7926) by the method initially developed by Datsenko K.A. and Wanner B.L.
  • E . coli strains 382 ⁇ argF ⁇ argI and 382 ⁇ argF ⁇ argI P tac7 - yajL are separately cultivated with shaking at 37°C for 18 h in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 ⁇ 200-mm test tubes and cultivated at 32°C for 48 h on a rotary shaker.
  • a solution of ninhydrin (2%) in acetone is used as a visualizing reagent.
  • a spot containing ornithine is cut out, ornithine is eluted with 0.5% water solution of CdCl 2 , and the amount of ornithine is estimated spectrophotometrically at 540 nm.
  • composition of the fermentation medium is as follows: Glucose 48.0 (NH 4 ) 2 SO 4 35.0 KH 2 PO 4 2.0 MgSO 4 ⁇ 7H 2 O 1.0 Thiamine-HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO 3 5.0
  • Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180°C for 2 h. The pH is adjusted to 7.0.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (8)

  1. Procédé de production d'un acide L-aminé comprenant :
    (i) la culture d'une bactérie produisant de l'acide L-aminé de la famille des Enterobacteriaceae dans un milieu de culture pour produire ledit acide L-aminé dans la bactérie ou le milieu de culture, ou les deux ; et,
    (ii) la collecte dudit acide L-aminé à partir de la bactérie ou du milieu de culture, ou les deux,
    dans lequel ladite bactérie a été modifiée pour surexprimer le gène yajL.
  2. Procédé selon la revendication 1, dans lequel ladite bactérie appartient au genre Escherichia.
  3. Procédé selon la revendication 2, dans lequel ladite bactérie est Escherichia coli.
  4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel ledit gène yajL est surexprimé en augmentant un nombre de copies du gène yajL ou en modifiant une séquence de régulation de l'expression du gène yajL de sorte que l'expression dudit gène soit accrue comparativement à une bactérie non modifiée.
  5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel ledit acide L-aminé est choisi dans le groupe constitué de la L-phénylalanine, du L-tryptophane, de la L-tyrosine, de la L-alanine, de la L-arginine, de la L-asparagine, de l'acide L-aspartique, de la L-citrulline, de la L-cystéine, de l'acide L-glutamique, de la L-glutamine, de la glycine, de la L-histidine, de la L-isoleucine, de la L-leucine, de la L-lysine, de la L-méthionine, de la L-ornithine, de la L-proline, de la L-sérine, de la L-thréonine et de la L-valine.
  6. Procédé selon la revendication 5, dans lequel ledit acide L-aminé est choisi dans le groupe constitué de la L-alanine, de la L-isoleucine, de la L-leucine, de la L-valine et de la L-phénylalanine.
  7. Procédé selon la revendication 5, dans lequel ledit acide L-aminé est la L-phénylalanine.
  8. Procédé selon la revendication 5, dans lequel ledit acide L-aminé est la L-valine.
EP15708898.0A 2014-02-14 2015-02-12 Procédé de production d'un acide l-aminé au moyen d'une bactérie de la famille des enterobacteriaceae dans laquelle le gène yajl est surexprimé Active EP3105338B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2014105547/10A RU2014105547A (ru) 2014-02-14 2014-02-14 СПОСОБ ПОЛУЧЕНИЯ L-АМИНОКИСЛОТ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИИ СЕМЕЙСТВА ENTEROBACTERIACEAE, ИМЕЮЩЕЙ СВЕРХЭКСПРЕССИРУЕМЫЙ ГЕН yajL
PCT/JP2015/054508 WO2015122544A1 (fr) 2014-02-14 2015-02-12 Procédé de production d'un acide l-aminé au moyen d'une bactérie de la famille des enterobacteriaceae dans laquelle le gène yajl est surexprimé

Publications (2)

Publication Number Publication Date
EP3105338A1 EP3105338A1 (fr) 2016-12-21
EP3105338B1 true EP3105338B1 (fr) 2019-01-30

Family

ID=52633554

Family Applications (1)

Application Number Title Priority Date Filing Date
EP15708898.0A Active EP3105338B1 (fr) 2014-02-14 2015-02-12 Procédé de production d'un acide l-aminé au moyen d'une bactérie de la famille des enterobacteriaceae dans laquelle le gène yajl est surexprimé

Country Status (7)

Country Link
US (1) US9493777B1 (fr)
EP (1) EP3105338B1 (fr)
JP (1) JP6447633B2 (fr)
CN (1) CN106029894B (fr)
BR (1) BR112016018257B1 (fr)
RU (1) RU2014105547A (fr)
WO (1) WO2015122544A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101866684B1 (ko) 2017-02-22 2018-07-04 대상 주식회사 L-세린을 생산하는 균주 및 이를 이용하는 l-세린의 제조 방법
JP2022521544A (ja) * 2019-02-22 2022-04-08 味の素株式会社 ｙｄｉＪ遺伝子を過剰発現する腸内細菌科に属する細菌を用いたＬ－アミノ酸の製造方法
CN110564660B (zh) * 2019-09-18 2023-03-21 苏州华赛生物工程技术有限公司 生产乳清酸的重组微生物及方法

Family Cites Families (89)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU875663A1 (ru) 1978-06-30 1982-09-15 Всесоюзный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Штаммы е.coLI ВНИИГенетика VL 334 @ N6 и ВНИИГенетика VL 334 @ N7-продуценты L-треонина и способ их получени
JPS561890A (en) 1979-06-15 1981-01-10 Ajinomoto Co Inc Preparation of l-phenylalanine by fermentation
JPS565099A (en) 1979-06-25 1981-01-20 Ajinomoto Co Inc Production of l-histidine through fermentation process and microorganism used therefor
JPS5618596A (en) 1979-07-23 1981-02-21 Ajinomoto Co Inc Production of l-lysine through fermentation process
JPS5672695A (en) 1979-11-15 1981-06-16 Ajinomoto Co Inc Preparation of l-leucine
JPS5685289A (en) 1979-12-13 1981-07-11 Ajinomoto Co Inc Preparation of l-valine by fermentation
JPS56144093A (en) 1980-04-14 1981-11-10 Ajinomoto Co Inc Preparation of l-proline by fermentation
JPS56144092A (en) 1980-04-14 1981-11-10 Ajinomoto Co Inc Preparation of l-methionine by fermentation
US4371614A (en) 1980-08-22 1983-02-01 Ajinomoto Co., Inc. E.Coli bacteria carrying recombinant plasmids and their use in the fermentative production of L-tryptophan
DE3127361A1 (de) 1981-07-08 1983-02-03 Schering Ag, 1000 Berlin Und 4619 Bergkamen Herstellung und anwendung von plasmiden mit genen fuer die biosynthese von l-prolin
JPH06102024B2 (ja) 1986-04-16 1994-12-14 味の素株式会社 新規プロモーター及び該プロモーターを用いた遺伝子発現方法
KR890003681B1 (ko) 1987-03-26 1989-09-30 주식회사 미원 미생물에 의한 l-페닐 알라닌의 제조방법
JP2536570B2 (ja) 1987-10-12 1996-09-18 味の素株式会社 発酵法によるｌ―イソロイシンの製造法
JP2748418B2 (ja) 1988-08-03 1998-05-06 味の素株式会社 組換えｄｎａ、該組換えｄｎａを有する微生物
US5175107A (en) 1988-10-25 1992-12-29 Ajinomoto Co., Inc. Bacterial strain of escherichia coli bkiim b-3996 as the producer of l-threonine
US5705371A (en) 1990-06-12 1998-01-06 Ajinomoto Co., Inc. Bacterial strain of escherichia coli BKIIM B-3996 as the producer of L-threonine
EP0488424B1 (fr) 1990-11-30 1997-03-05 Ajinomoto Co., Inc. Séquences d'ADN recombinantes codant pour des enzymes insensibles à l'inhibition de feedback, plasmides les comprenant, micro-organismes transformés utiles à la production d'acides aminés aromatiques, et procédé pour les préparer par fermentation
US5534421A (en) 1991-05-30 1996-07-09 Ajinomoto Co., Inc. Production of isoleucine by escherichia coli having isoleucine auxotrophy and no negative feedback inhibition of isoleucine production
BR9203053A (pt) 1991-08-07 1993-03-30 Ajinomoto Kk Processo para produzir acido l-glutamico pro fermentacao
JP3006926B2 (ja) 1991-09-04 2000-02-07 協和醗酵工業株式会社 発酵法によるｌ−スレオニンの製造法
JP3036930B2 (ja) 1991-11-11 2000-04-24 協和醗酵工業株式会社 発酵法によるｌ−イソロイシンの製造法
JP3151073B2 (ja) 1992-02-25 2001-04-03 協和醗酵工業株式会社 発酵法によるアミノ酸の製造法
RU2003677C1 (ru) 1992-03-30 1993-11-30 Всесоюзный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Штамм бактерий ESCHERICHIA COLI - продуцент L-гистидина
DE4232468A1 (de) 1992-09-28 1994-03-31 Consortium Elektrochem Ind Mikroorganismen für die Produktion von Tryptophan und Verfahren zu ihrer Herstellung
DE69219775T3 (de) 1992-10-14 2004-08-05 Ajinomoto Co., Inc. Neuartiges L-threoninproduzierendes Mikrobakterium und eine Herstellungsmethode für L-Threonin
DE69330518T2 (de) 1992-11-10 2002-05-08 Ajinomoto Kk Dna, die aspartokinase iii-mutanten kodiert und ihre verwendung zur herstellung von l-threonin durch fermentation
US5354672A (en) 1992-11-24 1994-10-11 Ian Fotheringham Materials and methods for hypersecretion of amino acids
AU687458B2 (en) 1993-10-28 1998-02-26 Ajinomoto Co., Inc. Process for producing substance
JPH07155184A (ja) 1993-12-08 1995-06-20 Ajinomoto Co Inc 発酵法によるｌ−リジンの製造法
JP3880636B2 (ja) 1994-01-10 2007-02-14 味の素株式会社 発酵法によるｌ−グルタミン酸の製造法
DE69534592T2 (de) 1994-03-04 2006-08-10 Ajinomoto Co., Inc. Verfahren für die herstellung von l-lysin
US5998178A (en) 1994-05-30 1999-12-07 Ajinomoto Co., Ltd. L-isoleucine-producing bacterium and method for preparing L-isoleucine through fermentation
JP3698758B2 (ja) 1994-06-30 2005-09-21 協和醗酵工業株式会社 発酵法によるｌ−ロイシンの製造法
DE69535674T2 (de) 1994-08-30 2009-01-02 Ajinomoto Co., Inc. Verfahren zur herstellung von l-valin und l-leucin
DE4440118C1 (de) 1994-11-11 1995-11-09 Forschungszentrum Juelich Gmbh Die Genexpression in coryneformen Bakterien regulierende DNA
CN101220366B (zh) 1994-12-09 2011-10-05 味之素株式会社 新的赖氨酸脱羧酶基因以及生产l-赖氨酸的方法
BR9610016A (pt) 1995-08-23 1999-07-06 Ajinomoto Kk Microorganismo e processo para a produção do ácido l-glutâmico por fermentação
JP4032441B2 (ja) 1995-08-30 2008-01-16 味の素株式会社 Ｌ−アミノ酸の製造方法
GB2304718B (en) 1995-09-05 2000-01-19 Degussa The production of tryptophan by the bacterium escherichia coli
DE19539952A1 (de) 1995-10-26 1997-04-30 Consortium Elektrochem Ind Verfahren zur Herstellung von O-Acetylserin, L-Cystein und L-Cystein-verwandten Produkten
JPH09285294A (ja) 1996-04-23 1997-11-04 Ajinomoto Co Inc 発酵法によるｌ−グルタミン酸の製造法
US5939307A (en) 1996-07-30 1999-08-17 The Archer-Daniels-Midland Company Strains of Escherichia coli, methods of preparing the same and use thereof in fermentation processes for l-threonine production
RU2119536C1 (ru) 1997-01-21 1998-09-27 Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Штамм escherichia coli - продуцент l-гистидина
DE19726083A1 (de) 1997-06-19 1998-12-24 Consortium Elektrochem Ind Mikroorganismen und Verfahren zur fermentativen Herstellung von L-Cystein, L-Cystin, N-Acetyl-Serin oder Thiazolidinderivaten
RU2140450C1 (ru) 1997-10-29 1999-10-27 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО "АГРИ") Штамм бактерий escherichia coli продуцент l-лейцина (варианты)
JP4151094B2 (ja) 1997-11-25 2008-09-17 味の素株式会社 Ｌ−システインの製造法
AU756507B2 (en) 1998-03-18 2003-01-16 Ajinomoto Co., Inc. L-glutamic acid-producing bacterium and method for producing L-glutamic acid
AU746542B2 (en) 1998-03-18 2002-05-02 Ajinomoto Co., Inc. L-glutamic acid-producing bacterium and method for producing L-glutamic acid
JP4294123B2 (ja) 1998-07-03 2009-07-08 協和発酵バイオ株式会社 ホスホリボシルピロリン酸を経由して生合成される代謝産物の製造法
JP4110641B2 (ja) 1998-11-17 2008-07-02 味の素株式会社 発酵法によるｌ−メチオニンの製造法
RU2175351C2 (ru) 1998-12-30 2001-10-27 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО "АГРИ") Фрагмент днк из escherichia coli, определяющий повышенную продукцию l-аминокислот (варианты), и способ получения l-аминокислот
RU2201454C2 (ru) 1999-07-09 2003-03-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Мутантная альфа-изопропилмалат синтаза (ipms), днк, кодирующая мутантную ipms, способ получения штамма escherichia coli, способ получения l-лейцина
JP4427878B2 (ja) 1999-08-20 2010-03-10 味の素株式会社 析出を伴う発酵法によるｌ−グルタミン酸の製造法
JP4245746B2 (ja) 1999-09-20 2009-04-02 協和発酵バイオ株式会社 発酵法によるアミノ酸の製造法
CA2319283A1 (fr) 1999-09-20 2001-03-20 Kuniki Kino Methode de production des acides amines-l
DE19949579C1 (de) 1999-10-14 2000-11-16 Consortium Elektrochem Ind Verfahren zur fermentativen Herstellung von L-Cystein oder L-Cystein-Derivaten
ES2313878T3 (es) 2000-01-21 2009-03-16 Ajinomoto Co., Inc. Procedimiento para la produccion de l-lisina.
RU2212447C2 (ru) 2000-04-26 2003-09-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Штамм escherichia coli - продуцент аминокислоты (варианты) и способ получения аминокислот (варианты)
RU2215783C2 (ru) 2001-05-15 2003-11-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото - Генетика" МУТАНТНАЯ N-АЦЕТИЛГЛУТАМАТ СИНТАЗА (ВАРИАНТЫ), ФРАГМЕНТ ДНК, ШТАММ БАКТЕРИИ Escherichia coli - ПРОДУЦЕНТ АРГИНИНА (ВАРИАНТЫ) И СПОСОБ ПОЛУЧЕНИЯ L-АРГИНИНА
JP4682454B2 (ja) 2000-06-28 2011-05-11 味の素株式会社 新規変異型ｎ−アセチルグルタミン酸合成酵素及びｌ−アルギニンの製造法
RU2207371C2 (ru) 2000-09-26 2003-06-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Способ получения l-аминокислот семейства l-глутаминовой кислоты, штамм бактерии escherichia coli - продуцент l-аминокислоты (варианты)
EP1172433B1 (fr) 2000-07-06 2006-06-14 Ajinomoto Co., Inc. Bactérie étant capable de produire de l'acide L-glutamique, de la L-proline et de la L-arginine, et procédé de préparation d'acide L-glutamique, de L-proline et de L-arginine
RU2208640C2 (ru) 2000-07-06 2003-07-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" СПОСОБ ПОЛУЧЕНИЯ L-АРГИНИНА, ШТАММ Escherichia coli - ПРОДУЦЕНТ L-АРГИНИНА
DE60219969T2 (de) 2001-02-13 2008-01-17 Ajinomoto Co., Inc. Methode zur Production von L-Aminosäuren mittels Bakterien der Gattung Escherichia
RU2215782C2 (ru) 2001-02-26 2003-11-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" СПОСОБ ПОЛУЧЕНИЯ L-АМИНОКИСЛОТ, ШТАММ Escherichia coli - ПРОДУЦЕНТ L-АМИНОКИСЛОТЫ (ВАРИАНТЫ)
RU2209248C2 (ru) 2001-06-26 2003-07-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Способ получения l-метионина, штамм бактерии escherichia coli вкпм в-8125 - продуцент l-метионина
RU2264459C2 (ru) 2001-08-03 2005-11-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Новая мутантная карбамоилфосфатсинтетаза и способ продукции соединений - производных карбамоилфосфата
MXPA04004874A (es) * 2001-11-23 2004-09-03 Ajinomoto Kk Procedimiento para producir l-aminoacido utilizando escherichia.
RU2229513C2 (ru) 2001-11-23 2004-05-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Способ получения l-аминокислот, штамм escherichia coli - продуцент l-аминокислоты (варианты)
KR100459758B1 (ko) 2002-05-15 2004-12-03 씨제이 주식회사 이소루이신 조절이 해제된 트레오닌 오페론 염기서열 및그를 포함하는 형질전환 세포를 이용한 ｌ-트레오닌의생산방법
DE10314618A1 (de) * 2003-04-01 2004-10-14 Degussa Ag Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von Stämmen der Familie Enterobacteriaceae
RU2279477C2 (ru) 2003-12-05 2006-07-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) Мутантная серинацетилтрансфераза, фрагмент днк, кодирующий мутантную серинацетилтрансферазу (варианты), бактерия, принадлежащая к роду escherichia, - продуцент l-цистеина, и способ продукции l-цистеина
EP1649403A2 (fr) 2003-07-29 2006-04-26 Ajinomoto Co., Inc. Procede permettant de detecter la production d'une substance influen ant les flux metaboliques
JP4380305B2 (ja) 2003-11-21 2009-12-09 味の素株式会社 発酵法によるｌ−アミノ酸の製造法
KR100786987B1 (ko) 2004-01-30 2007-12-18 아지노모토 가부시키가이샤 Ｌ-아미노산 생산 미생물 및 ｌ-아미노산 생산 방법
WO2005103275A1 (fr) 2004-04-26 2005-11-03 Ajinomoto Co., Ltd. Procédé de fabrication de l-tryptophane selon le procédé de fermentation
US7915018B2 (en) 2004-10-22 2011-03-29 Ajinomoto Co., Inc. Method for producing L-amino acids using bacteria of the Enterobacteriaceae family
EP1838726A1 (fr) 2005-01-18 2007-10-03 Ajinomoto Co., Inc. Micro-organismes produisant un acide amine l et procede permettant de produire un acide amine l
JP2009095237A (ja) 2006-02-02 2009-05-07 Ajinomoto Co Inc Ｌ−アミノ酸の製造法
JP2009060791A (ja) 2006-03-30 2009-03-26 Ajinomoto Co Inc Ｌ−アミノ酸生産菌及びｌ−アミノ酸の製造法
RU2355763C2 (ru) 2006-09-13 2009-05-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) Мутантная ацетолактатсинтаза и способ продукции разветвленных l-аминокислот
JP2010041920A (ja) 2006-12-19 2010-02-25 Ajinomoto Co Inc Ｌ−アミノ酸の製造法
JP2010130899A (ja) 2007-03-14 2010-06-17 Ajinomoto Co Inc Ｌ−グルタミン酸系アミノ酸生産微生物及びアミノ酸の製造法
RU2395579C2 (ru) 2007-12-21 2010-07-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) СПОСОБ ПОЛУЧЕНИЯ L-АМИНОКИСЛОТЫ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИИ, ПРИНАДЛЕЖАЩЕЙ К РОДУ Escherichia
RU2396336C2 (ru) 2007-09-27 2010-08-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) СПОСОБ ПОЛУЧЕНИЯ АМИНОКИСЛОТ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИИ, ПРИНАДЛЕЖАЩЕЙ К РОДУ Escherichia
RU2009136544A (ru) 2009-10-05 2011-04-10 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) Способ получения l-цистеина с использованием бактерии семейства enterobacteriaceae
RU2460793C2 (ru) 2010-01-15 2012-09-10 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) Способ получения l-аминокислот с использованием бактерий семейства enterobacteriaceae
RU2012112651A (ru) 2012-04-02 2013-10-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") САМОИНДУЦИРУЕМАЯ ЭКСПРЕССИОННАЯ СИСТЕМА И ЕЕ ПРИМЕНЕНИЕ ДЛЯ ПОЛУЧЕНИЯ ПОЛЕЗНЫХ МЕТАБОЛИТОВ С ПОМОЩЬЮ БАКТЕРИИ СЕМЕЙСТВА Enterobacteriaceae
RU2013118637A (ru) 2013-04-23 2014-10-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") СПОСОБ ПОЛУЧЕНИЯ L-АМИНОКИСЛОТ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИИ СЕМЕЙСТВА ENTEROBACTERIACEAE, В КОТОРОЙ РАЗРЕГУЛИРОВАН ГЕН yjjK

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
JP2017505631A (ja) 2017-02-23
RU2014105547A (ru) 2015-08-20
BR112016018257A2 (pt) 2018-10-23
WO2015122544A1 (fr) 2015-08-20
JP6447633B2 (ja) 2019-01-09
US20160340681A1 (en) 2016-11-24
BR112016018257B1 (pt) 2022-01-25
CN106029894B (zh) 2020-09-01
EP3105338A1 (fr) 2016-12-21
CN106029894A (zh) 2016-10-12
CN106029894A8 (zh) 2017-12-01
US9493777B1 (en) 2016-11-15

Similar Documents

Publication Publication Date Title
EP1899452B1 (fr) Procede de production d'un acide l amine au moyen d'une bacterie de la famille des enterobacteriaceae a expression renforcee de l' operon fucpikur
US9873898B2 (en) Method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having attenuated expression of a phosphate transporter-encoding gene
US9175319B2 (en) Method for producing an L-amino acid using a bacterium of the family enterobacteriaceae having attenuated expression of the yjjK gene
US20090269819A1 (en) METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF ANY OF THE cynT, cynS, cynX OR cynR GENE OR COMBINATION THEREOF
EP2007873B1 (fr) PROCÉDÉ DE PRODUCTION D'UN ACIDE L-AMINÉ PAR UNE BACTÉRIE DE LA FAMILLE DES ENTEROBACTÉRIACÉE AVEC EXPRESSION ATTENUÉE DE L'AGRÉGAT sfmACDFH-fimZ OU DU GÈNE fimZ
US10392638B2 (en) Method for producing L-amino acids using a bacterium of the family enterobacteriaceae overexpressing a gene encoding an iron exporter
EP3098319B1 (fr) Procédé de production d'un acide l-aminé au moyen d'une bactérie de la famille des enterobacteriaceae présentant une expression atténuée du gène gsha
EP2559754B1 (fr) Procédé de production d'un acide l-aminé utilisant une bactérie de la famille des entérobactéries à expression améliorée des gènes de formation des flagelles et de cascade motilitée
US9493777B1 (en) Method for producing an L-amino acid using a bacterium of the family enterobacteriaceae having overexpressed the yajL gene
EP1929027B1 (fr) UNE MÉTHODE POUR PRODUIRE UN ACIDE L-AMINÉ AU MOYEN D' UNE BACTÉRIE DE LA FAMILLE DES ENTÉROBACTÉRIACEAE À EXPRESSION ATTÉNUÉE DU GÈNE ybiV
US9840725B2 (en) Method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having a disrupted putrescine degradation pathway
WO2015030019A1 (fr) Procédé de production d'un acide l-aminé par utilisation d'une bactérie de la famille des enterobacteriaceae ayant une expression atténuée de la batterie de gènes znuacb

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20160914

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

RIC1 Information provided on ipc code assigned before grant

Ipc: C12P 13/06 20060101ALI20180702BHEP

Ipc: C12P 13/24 20060101ALI20180702BHEP

Ipc: C12P 13/04 20060101AFI20180702BHEP

Ipc: C12P 13/10 20060101ALI20180702BHEP

Ipc: C12P 13/12 20060101ALI20180702BHEP

Ipc: C12N 15/70 20060101ALI20180702BHEP

Ipc: C12P 13/14 20060101ALI20180702BHEP

Ipc: C12P 13/22 20060101ALI20180702BHEP

Ipc: C12P 13/08 20060101ALI20180702BHEP

INTG Intention to grant announced

Effective date: 20180802

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

RIN1 Information on inventor provided before grant (corrected)

Inventor name: STOYNOVA, NATALIA VIKTOROVNA

Inventor name: DOROSHENKO, VERA GEORGIEVNA

Inventor name: EREMINA, NATALIA SERGEEVNA

Inventor name: SAMSONOV, VALERY VASILIEVICH

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1093294

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190215

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602015023969

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190430

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190530

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1093294

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190430

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190501

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190530

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190212

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602015023969

Country of ref document: DE

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20190228

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190228

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190228

26N No opposition filed

Effective date: 20191031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190212

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190228

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190212

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20150212

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20220118

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190130

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SK

Payment date: 20230113

Year of fee payment: 9

Ref country code: DE

Payment date: 20221229

Year of fee payment: 9

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230512

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20240108

Year of fee payment: 10

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20231228

Year of fee payment: 10

Ref country code: GB

Payment date: 20240108

Year of fee payment: 10

Ref country code: SK

Payment date: 20240115

Year of fee payment: 10